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Objective: To explore the mesenchymal stem cells in the bone marrow (bone marrow mesenchymal stem cells, BMSCs) isolated and cultured in vitro and induced chondrocyte differentiation factors; explore natural biomaterials silk fibroin (silk fibroin, SF) and chitosan (Chitosan, CS) prepared by mixing the physical and chemical properties of the SF-CS scaffold material and biocompatibility. Method 1, extract of rabbit bone marrow fluid separation in the purification of bone marrow fluid mesenchymal stem cells, MTT assay was used to detect 1-5 generation cell growth activity. Passaged to take amplification 3rd generation of BMSCs experimental group and the control group, the experimental group HG-DMEM culture medium with 10% fetal bovine serum added transforming growth factor-β1, dexamethasone, and ascorbic acid induced by the control group with 10 % FBS HG-DMEM culture medium natural differentiation, induction of 4 days, 7 days, 14 days, respectively, with toluidine blue staining cells glycosaminoglycan expression, the expression of type II collagen immunohistochemical staining cells . 2, silk degumming, dissolved and purified to obtain the appropriate concentration of silk fibroin solution, mixed in different proportions of the silk fibroin solution with chitosan solution into Groups A, B, C and D, vacuum freeze-drying prepared SF-CS scaffold, using 1 - ethyl-3-(3 - dimethyl-aminopropyl) carbodiimide (EDC) / N-hydroxysuccinimide (NHS) / do Lin - ethanesulfonic acid (MES) as a crosslinker using the liquid alternative method detects SF-CS scaffolds porosity, scanning electron microscopy the ultrastructure, Hot Water failure detected the Hot Water loss rate before and after each group, the crosslinking; induced cartilage cells and the scaffold extracts mixed culture, MTT assay was used to detect the toxic effects of stent cells; induced chondrocyte differentiation cell vaccination the groups material is divided into group A, group B, group C, D groups of materials and pure culture plate group, at each time point with cell counting and MTT method to check the rate of cell attachment and cell viability. Results: 1, 1-3 passages of cells living cell rate is about 96% of cells 4-5 cells in living cells was significantly lower, BMSCs directed inducing experimental group cells stained with toluidine blue metachromatic, and type II collagen Immunohistochemical staining positive rate compared with the control group, a significant difference. 2, SF-CS bracket has an irregular porous structure, traffic Kongfeng Fu, in each group the average pore diameter of between 137-164μm, a porosity in the range of from 83-92%, after the crosslinking agent after processing Material Hot Water loss rate significantly reduced, on average around 23%. MTT assay scaffold for cell toxicity results analysis: the material is non-toxic to cells; cell count detection rate of cell adhesion and MTT assay cell activity, between each set of materials and flat-culture group twenty-two comparison, the difference was statistically significant ( P lt; 0.05), B group (SF: CS = 50:50), cell adhesion rate and cell activity is higher than the other groups. Conclusion: 1, rabbit 3rd generation BMSCS has stable growth, proliferation speed and ability cloning is ideal for cartilage tissue engineering seed cells, and its cartilage cells induced to differentiate in vitro to seven days II collagen expression most obvious. 2, SF-CS scaffold has a good biological characteristics, the mixing ratio of the two impact SF-CS biological activity, the mixing ratio of 50:50 with the best biological activity, can be used as carrier materials for cartilage tissue engineering security applications.
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